The invention relates to determining the presence of chromosomal abnormalities, such as abnormal numbers of specific chromosomes, in cells.
All cells contain DNA comprising the genetic information necessary to control a cell's biologic function. DNA is made up of two linear strands of four different chemical building blocks or nucleotides arranged in specific sequences which are translated by mechanisms in cells to control the manufacture of specific proteins such as enzymes. The total of the some 100,000 genes in humans, each of which codes for one specific protein, constitute the genome of an individual. These genes are organized into rod-like chromosomes which can be visualized microscopically for only a brief time in a cell's life cycle, during the so-called metaphase, which is just prior to cell division. In humans, each cell contains 46 chromosomes, 23 of which are contributed by each parent. As a result, many genes occur on two different chromosomes and are located at two separated positions in the interphase cell nucleus.
Medical research has shown links between flaws in specific genes or chromosomes and certain diseases. Of major importance are gene modifications causing cancer or birth defects such as Down's syndrome, or a predisposition to certain cancers. Such genetic modification may take any of the following forms: 1) aneuploidy, an abnormal number of one of the 23 chromosomes; 2) translocations, genetic material moved to a wrong chromosome; 3) rearrangement mutations, genetic material moved to the wrong place on a chromosome; 4) amplifications, an abnormal number of copies of a specific gene; 5) deletion mutations, a specific gene segment is missing; and 6) point mutations, altered nucleotides in a gene sequence. Of particular interest are mutations of genes which may enhance or suppress tumor growth, the so-called oncogenes and tumor suppressor genes.
It is important to identify such genetic modifications to diagnose or predict certain diseases. For example, chromosome banding techniques are widely used to identify numerical and/or structural chromosome aberrations in tumor and prenatal diagnosis. However, the interpretation of the banding patterns requires skilled technicians, is often complicated by imperfect banding, chromosome condensation, and limited numbers of metaphases, and is difficult, e.g., in cases of highly aneuploid tumors with extensive structural changes.
An alternative method to detect chromosomal aberrations is an in situ hybridization technique, which uses chromosome-specific probes to analyze nuclear DNA directly when the cells are in interphase. A variation of this method, called fluorescent in situ hybridization (FISH), also involves a nucleic acid probe with a defined nucleotide sequence that preferentially hybridizes with a specific complementary nucleotide sequence of DNA, or target DNA, on one or more chromosomes in a cell. The target nucleotide sequence may be unique or repetitive, as long as it can be used to distinguish one or more specific chromosomes. In the FISH technique, the probe is marked with a fluorescent label so that cells with the target DNA sequence(s), to which the marked probes hybridize, can be detected microscopically. Each chromosome containing the target DNA sequence(s), and hence the marked probe, will emit a fluorescent signal or spot in every cell.
For example, a cell sample allowed to hybridize with a fluorescently labeled DNA probe that hybridizes to a specific target nucleotide sequence on chromosome number 21 will show two fluorescent spots in each cell from a normal person, and three spots in each cell from a Down's syndrome patient, because these patients have an extra chromosome number 21. Probes specific for chromosome 21 are well known. See, e.g., Pinkel et al., P.N.A.S., USA, 85:9138-9142 (1988), which is incorporated herein by reference.
The six different genetic abnormalities described above are detected by the FISH technique as follows. Aneuploidy is determined by counting spots per cell using a DNA probe specific to one chromosome. Translocations and rearrangements are determined by using DNA probes covering the translocation or rearrangement and a neighboring sequence and determining whether the spots from each sequence are separated or concentric. Amplification, deletion, and point mutations are determined by quantifying the fluorescence from spots using FISH for a specific target nucleotide sequence.
The FISH technique can be used for a variety of diagnostic and screening tests. For example, it can be used in conjunction with techniques such as amniocentesis and chorionic villus sampling (CVS) to screen fetuses to determine whether the baby will have a serious birth defect such as Down's syndrome. Both amniocentesis and CVS are associated with the risk of miscarriage, which may be minimized by the FISH technique. This risk is estimated at 1.0% to 2.0% for CVS and at 0.5% for amniocentesis. It may soon be possible to sidestep that risk entirely by obtaining fetal cells from the mother's blood, so that only a blood sample rather than an umbilical cord sample is required.
To apply the FISH technique as a prenatal screening tool, sets of DNA probes may be used that hybridize to regions of five different chromosomes, e.g., 21, 18, 13, X, and Y, which together account for 90% to 95% of all birth defects related to chromosomal abnormalities.
There are also FISH tests proposed for cancer screening, diagnosis, prognosis, and treatment monitoring in which the presence or the absence of specific gene sequences must be determined in patient cell samples. Such screening and diagnosis currently requires technicians to visually count fluorescent spots in each cell under a microscope. However, such manual microscopic visualization is quite laborious and is therefore not currently performed on a routine basis.